PROJECT SUMMARY Every year, ~40,000 women in the US who had been successfully treated for primary breast cancer nonetheless have metastatic recurrence. Metastasis requires four key steps: 1) tumor cells leave the tumor; 2) tumor cells enter a new tissue; 3) disseminated tumor cells (DTCs) re-initiate proliferation; and 4) an inflammatory microenvironment is established to support the growing metastasis. Steps 1-2 often happen before the cancer is diagnosed and are rarely amenable to intervention, but we may be able to target steps 3-4 to reduce the occurrence of metastasis. To accomplish this long-term goal, we must determine a) how the quiescent DTCs re-initiate proliferation and b) how the metastasis-supporting inflammatory microenvironment is established. Our research on neutrophil extracellular traps (NETs) has provided novel insights into how these processes can occur. NETs consist of meshes of genomic DNA with ~40 associated proteins, and they are released by neutrophils to the extracellular space in response to infections and inflammation. In mouse models, we found that lung inflammation, induced by tobacco smoke or bacterial lipopolysaccharide, triggered quiescent DTCs to re-initiate proliferation, causing lethal metastases. Using intravital imaging, we found that DTCs were surrounded by NETs after lung inflammation. We discovered that proteases on NETs cleave laminin, a basement membrane protein. This cleavage generated a laminin epitope that activated integrin receptors, triggering quiescent DTCs to proliferate. To interfere with this mechanism, we developed an antibody against NET-cleaved laminin (ChiAb28). ChiAb28 prevented metastasis in over half of the mice and reduced metastasis in the remaining mice. Thus, NET-associated proteases drive step 3 of the metastatic process. In parallel, our new data show that other NET-associated proteins activate macrophages to secrete interleukin (IL)-1?, which in turn induces more NETs and supports the metastases. This NET/macrophage feedback loop may create an inflammatory microenvironment that supports the metastasis (step 4). Our data have led us to hypothesize that NETs trigger metastases from quiescent DTCs by a) cleaving laminin to re-initiate proliferation and b) driving a feedback loop that causes local inflammation. How NETs cleave laminin, however, is still unclear, and we will determine these mechanisms in Aim 1. How the feedback loop between NETs and macrophages via IL-1? secretion maintain the growth of the metastasis will be determined in Aim 2. Finally, we will combine our unique expertise in NET biology, the tumor microenvironment, and intravital imaging to investigate combination treatments with ChiAb28 and either chemotherapy or IL-1? blocking antibodies as new therapeutic approaches to prevent metastasis in Aim 3. This project will determine the mechanisms that are responsible for NETs' ability to drive the transition from quiescent to proliferating DTCs and further to lethal metastases. New knowledge of how NETs promote metastasis may ultimately allow the development of NET-targeting strategies to prevent metastasis.